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Science Highlight: June 27, 2016

NIBIB-funded approach could advance drug development

In 2011, researchers supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB), part of the National Institutes of Health, developed a technique, called phage-assisted continuous evolution (or PACE), that rapidly generates proteins with new, sought-after properties and therapeutic potential. Originally conceived as a tool for pharmaceutical development, the researchers now have shown its potential in protecting crops from insects.

The cabbage looper, whose larvae is depicted on a potato leaf, has developed resistance to a soil bacterium called Bacillus thuringiensis (Bt) and is frequently studied for insect resistance to Bt toxins. Photo courtesy of USDA, by Peggy Greb.

“PACE has proven to be an efficient tool for evolving protein variants with new and useful functions,” said David Rampulla, Ph.D., Director of the NIBIB program in Synthetic Biology for Technology Development. “Adapting PACE to the problem of resistant insects in crops broadens the technique, enabling researchers to create new binding capabilities for proteins that could have a significant impact on pharmaceutical development.”

Proteins typically evolve gradually in response to an environmental selection pressure. PACE puts evolution in overdrive by taking advantage of how viruses that infect bacteria, called bacteriophages or phages, use the bacterial proteins to quickly replicate themselves and their genes.

“PACE enables us to perform protein evolution at a speed that’s about 100 times faster than methods previously used in the laboratory,” said David Liu, Ph.D., a Howard Hughes Medical Institute Investigator at Harvard University. Liu is senior author of a new study published online May 5, 2016 in Nature, in which his team used PACE to develop a protein insecticide with the potential to be rapidly updated to avert insect resistance.

The technique manipulates the phage replication process so that a particular desired feature—such as binding a target protein— becomes a requirement for phage replication, so only the phage that encode proteins with the desired characteristic survive. PACE also greatly boosts the phage mutation rate, which enables much more rapid changes in protein sequences during evolution.

In this new study, the researchers turned to the problem of agricultural pests. Insect resistance to biological insecticides is a serious and growing problem for agricultural production. Crops engineered to contain insecticidal proteins from B. thuringiensis (Bt toxins) are widely used around the world as safe and cost-effective alternatives to spraying pesticides. But as more insects acquire resistance to Bt toxins, the crops become vulnerable to infestation and ruin. The new research used PACE to rapidly create mutated toxins that sidestep current resistance and provide a way to stay ahead of insect pest resistance in the future.

Overview of the phage-assisted continuous evolution (PACE) scheme. Phage that make proteins capable of binding to the target protein are retained (selected for) because of rapid growth, while those that do not make a desired binding protein grow slowly and are eliminated from the system.

To create the mutated Bt toxin, the researchers used PACE to make billions of Bt toxin variants, requiring that the variants bind to a new protein in the insect gut. The idea was to create a mutated version of the toxic protein that had the ability to bind to a receptor that natural Bt toxin doesn’t normally interact with, killing the bug in a novel way. In just a few weeks, with very little researcher intervention, the technique created multiple variants of the toxin that kill bugs that had acquired resistance to the natural Bt toxin.

The technology could give farmers the option to simultaneously use multiple insecticides that each target a different insect protein, making it harder for pests to achieve resistance. Alternatively, PACE could whip up new variants as needed, whenever resistance starts to spread.

Liu says that this application of PACE, which seeks to improve the ability for crop producers to feed people, is a substantial departure from what his laboratory normally does. “But the nature of the science is still the same,” Liu said. “We are trying to evolve molecules that have properties that will dramatically enhance their usefulness.”

“Next we hope to use the protein-binding PACE system to evolve proteins that have enhanced therapeutic potential by virtue of their new protein binding activity or specificity,” Liu said. “A protein’s binding to another protein is a key step in many biological processes involved in human disease.”

Scientists from Harvard University in Cambridge, Massachusetts; Monsanto Company in Cambridge, Massachusetts and Chesterfield, Missouri; and Cornell University in Geneva, New York performed the research.

The work was supported through grant EB022376 from NIBIB. Additional support was provided by the Defense Advanced Research Projects Agency, the Howard Hughes Medical Institute, the US Department of Agriculture, and the National Science Foundation.